Control sperm
The 3 adapters were ligated using SRBC barcode adapters for each sample, additionally, 18-mer and 30-mer markers were ligated and used as control for the ligation process and markers for the product correct size. The 3-ligated small RNAs were size-selected using 15% denaturing PAGE and the 3 ligated RNAs ranging from 18 -30 mer were cut out. RNAs were and the elute 3-ligated small RNAs were eluted in 5 linker mix containing 5 adaptor. The 3-ligated RNAs + 5 adapter were denatured for 5 min at 70C, cooled on ice immediately, ligated with T4 RNA ligase and incubated at 16 degrees overnight.  Ligated small RNAs were purified by using MBS beads, briefly: MBS buffer, MBS bead slurry (beads + buffer), mixed by vortexing, added isopropanol and incubated at room temperature. Beads were separated on magnet and the supernatant was removed, after several washes with ethanol the RNA was eluted with ultrapure water and transferred into PCR strip. For reverse transcription, small-RNAseq RT primer to each sample was used and a negative control without reverse transcriptase was included, Superscript II reverse transcriptase was used to obtain the synthesis of the first strand. To amplify cDNA libraries, KAPA HiFi Real Time Library Amplification Kit was used; PCR were performed using TruSeq Universal Adapter primer (Solexa_PCR_fwd) and TruSeq Index reverse primers (Solexa_IDX_rev), this latter includes barcodes assigned to each different sample. Briefly: master mix was added and TruSeq Index reverse primer were added to PCR strips containing cDNAs; then KAPA HiFi HS RM and Truseq Universal Adapter primer were added to the mix. The cycling program was: Denaturation at 98C for 45 sec; 20 cycles of 98C for 15 sec, 65C for 30 sec, 72C for 30 sec, 72C for 10 sec; and a final extension at 72C for 1 min. The amplified cDNA was purified by using 3% Low-Range Ultra Agarose gel using GeneRuler 50bp DNA Ladder as molecular marker. DNA band between 150-200 bp were excised using a clean scalpel blade and put into a clean 15ml Falcon tube; the DNA was purified and used for sequencing